Funding for this work was provided by: AG007218 and NIH F32 AG031606.
Images were generated in the Core Optical Imaging Facility, which is supported by UTHSCSA, NIH-NCI P30 CA54174 (CTRC at UTHSCSA) and NIH-NIA P01AG19316.

注：すべての動物の手順は、テキサス大学健康科学センターのサンアントニオの施設内動物管理使用委員会によって承認され、ARRIVEガイドラインと一致しました。 1.皮質の準備のために麻酔<ol><li>麻酔を誘導するために、酸素と混合して2〜3％のイソフルランで誘導チャンバ内にマウスを置きます。マウスが誘導されると呼吸速度の低下を観察します。マウスはノーズコーンに移動する準備ができているかどうかを決定するために、マウスの足をつまみます。注：麻酔レベルは、任意のin vivoでの調製における重要なステップであるとケアがグローバル虚血の原因となるレベルを誘発しないように注意してください。 </li><li>マウスが十分に麻酔されると、手術/イメージングプラットフォームに動物を転送し、ノーズコーンにマウスの鼻を配置し、麻酔状態を維持するために、1から1.2パーセントのイソフルランを適用します。マウスは、温度共に横たわっていることを確認してくださいntrolled加熱パッドは、残りの手順を通して、体温（37℃+/- 0.5℃）を維持します。麻酔下ながら乾燥を防ぐために、目の上の獣医軟膏を置きます。 </li><li>システムに付属の尾や足のクリップを使用して、パルス酸素濃度計システムを使用して、マウスの生理機能を監視します。呼吸数が50〜65呼吸/分の間で維持されることを確認してください。 300から450 BPMと酸素飽和度は、動物の長期生存を確保するために、97から98までパーセントの間で維持される間に心拍数が残っていることを確認してください。 </li><li>マウスが十分に麻酔されると、電気バリカンを用いて頭蓋の上に髪を剃るエタノール綿棒に続く残留髪とベタジンできれいにし、削除します。無菌手術用環境を確保するために3回にこの手順を繰り返します。 </li></ol> 2.手術手順<ol><li>頭皮が完全に洗浄され、坊主で、頭蓋fissuを明らかにするために、マウスの頭皮に、5mmの切開を作りますブレグマを検索する解像度と。 </li><li>頭蓋の上にある残りの筋膜を除去するために滅菌綿棒を使用してください。 </li><li>ブレグマの定位座標を用いて、頭頂葉皮質の上にある骨に組織接着剤を使用したカスタムステンレス製リング（ 図1）接着剤：横-1〜-3ミリ、2-4ミリメートル。注：接着剤は、典型的には、骨の上にリングを配置した後、2分以内に設定します。 </li><li>マウスを安定させ、画像形成中の移動を防止するために、定位ホルダー（ 図2）環を貼付。 </li><li>外科用グレードの解剖顕微鏡下で、ゆっくりとそれが開けられるように領域レベルを維持することを確認すること速度制御DREMELのようなツール（Meisinger 3.9ミリメートルドリルビット）を使用して、頭蓋中で1〜2ミリのセクションをドリル。ジグザグパターンを使用してこれを達成します。熱を避けるために構築、低へのドリル速度を設定し、頻繁に休憩を取ります。 </li><li>頭蓋頭蓋骨は、外観に登るとなると、間伐を続けます頭蓋頭蓋骨の薄層の円滑な除去を容易にするために薄くなった面レベルを維持するために、同じジグザグパターンを利用して手術用メスの刃を使用して頭蓋。メスの刃の先端を使用すると、一度に骨の薄い層を除去するための光の圧力で小さなリニアストロークを行います。血管系は、解剖顕微鏡を通してはっきりと表示されるまでこれを続けます。 </li><li>実験者の穿刺を介して、または間引き処理中に頭蓋骨を破壊した場合、基礎となる皮質への可能性損傷に動物を安楽死させます。 注：マウスの​​頭蓋骨は、約300ミクロンの厚さであり、2つの薄い緻密骨の層（1外部と一つの内部層）とコンパクトな骨の2つの層の間に挟まれた海綿骨の層で構成されています。外部コンパクト骨の層および海綿骨の大部分が残っているコンパクトな骨のおおよそ50μmの層が得られ、5mmの掘削領域内に削除されます（ 図2B参照します ）。 Visualiz血管系のationが最終そのまま薄く頭蓋骨が約50μmの厚さであることを保証します。この厚さまで薄くする際の頭蓋骨はまだそのために存在します。 </li></ol> 3.顕微鏡セットアップ<ol><li>客観的なインバータを有している倒立顕微鏡システム（従来、共焦点または二光子システム）を使用します。注：これは、標準的な正立顕微鏡を利用することも可能です。制限要因は、ステージと目標との間の空間になります。ステージへの変更は、このセットアップを実行する必要があるかもしれません。 </li><li>脇顕微鏡のベースに位置してカスタムメイドのステージに外科的/撮影台を固定します。注：プラットフォームは、対物下外科手術/撮影台の垂直運動を可能にするために、実験室ジャッキを使用して行われます。実験用ジャッキは、4つの円筒状のポールに固定されたプレートに取り付けられています。 （ 図2を参照してください）。 </li><li> 20Xを含む客観的なインバータを置き頭蓋窓の上の目的。顕微鏡の接眼レンズを通して見ることにより、頭蓋窓を見つけ、撮影領域に目標を配置するために外部光源を使用してください。注：撮像領域は、脈管構造の存在によって示されます。 </li><li>水ベースの目的のために、人工脳脊髄液（aCSFの）を使用します（; 30のKCl、130mMの塩化ナトリウムを12mMのKH 2 PO 4、200mMの炭酸水素ナトリウム、30mMのHEPES、100 mMグルコース）培地として、潜在的に撮影時の頭蓋腔への漏出（ 図3）。 </li></ol> 4.ローズベンガル染料準備、管理および脳卒中の誘導<ol><li>人工脳脊髄液（aCSFの）中のローズベンガルの新鮮な20 mg / mlの溶液を調製します。フィルタリングし、投与前に滅菌します。それが混合された後にローズベンガルを再利用したり、保管しないでください。各実験のために新鮮なソリューションを行います。 </li><li>ローズベンガルの一方で、0.1mlの尾静脈注射を与えます溶液の適切な注入を保証するために、561 nmのレーザーで頭蓋窓を缶詰。注：ローズベンガルは、脳の血管系内注射後5秒以内に可視化されます。容器全体は、ローズベンガルで満たされるべきです。 </li><li>ローズベンガル色素の十分な注射後特定の病変体積の再現性を保証するために、血管の直径（40〜80ミクロン）に基づいて、血栓症の適切な容器を選択します。血流の方向を見て、動脈と静脈を区別：動脈は小径血管に大きな直径から移動し、静脈はより大きな直径の血管に小さいから移動します。ローズベンガルが注入されると、これは、容易に可視化することによって達成されます。 </li><li>次のように顕微鏡の設定を変更します。 <ol><li>滞留時間を増加させます。注：これは、利用される顕微鏡システムに依存して変化します。 </li><li> 100％にレーザーパワーを大きくしてください。 </li><li>使用して1フレーム/秒で時系列画像を収集最大スキャン速度。 </li></ol></li><li>安定した凝血塊が血管内に形成されるまでマウスをスキャンします。注：この一般的に連続スキャンの5分以内に達成される（ 図4を参照してください）。 </li><li>ローズベンガルを用いた血栓形成の誘導後、バック解剖顕微鏡の撮像領域からマウスを取り外します。慎重に頭蓋頭蓋骨からステンレス製のリングを取り外します。出血のための頭蓋窓を調べます。出血が発生した場合は、実験を終了します。 </li><li>頭蓋骨の上に切開部を閉じるために、6.0モノフィラメント縫合糸を使用してください。感染を防ぐために、縫合線に沿って抗生物質軟膏を置きます。 Buprenex（0.05ミリグラム/ kg）の疼痛管理のために3日間皮下12時間毎に注入します。 </li><li>完全に覚醒し、自由に移動するまで麻酔から除去した後の回復室にマウスを返します。 </li><li>後で、さらなる調査のためにきれいなケージにマウスを返します。 </li></ol> 5。その後の日に縦イメージング<ol><li>その後の日後に光血栓の縦撮影を行うには、次の方法を採用しています。 <ol><li>方法の第1節で説明したように、マウスを麻酔。 </li><li>十分な麻酔の際に、以前に掘削撮像視野を覆う皮膚を再度オープンするために、任意の残留縫合糸を除去することにより、頭皮を再オープンします。 </li><li>頭蓋の上にある残りの筋膜を除去するために滅菌綿棒を使用してください。 </li><li>前回の撮像視野を覆う骨に組織接着剤を使用したカスタムステンレス製リング（ 図1）を接着。 </li><li>マウスを安定させ、画像形成中の移動を防止するために、定位ホルダー（ 図2）環を貼付。 </li><li>以前に薄く頭蓋骨の基礎となる血管系を探します。以前に誘導された血餅の存在を確認するためにFITCデキストランの尾静脈注射を使用してください。 </li><li>内を閉じるために6.0モノフィラメント縫合糸を使用頭蓋骨の上cision。感染を防ぐために、縫合線に沿って抗生物質軟膏を置きます。 Buprenex（約0.05mg / kg）を、疼痛管理のために3日間皮下12時間毎に注入します。 </li><li>完全に覚醒し、自由に移動するまで麻酔から除去した後の回復室にマウスを返します。 </li></ol></li></ol>脳卒中の誘導の6検証（死後） <ol><li>研究の終わりに、 図5に示すように、2,3,5-トリフェニルテトラゾリウムクロリド（TTC）染色を使用して、ストローク量を確認する。完全な方法は、12に見出すことができます。 </li></ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+fluorescence+imaging+with+high-resolution+microlenses.">In vivo fluorescence imaging with high-resolution microlenses.</a> Nature Methods. 6, 511-512 (2009).</li></ol>

著者は、彼らが競合する金融利害関係がないことを宣言します。

動物からヒトへの適用に実験的なストローク病態生理を変換する機能は、障害に悩まされています。しかし、このような光血栓モデルなどの動物モデルの使用は、改良されたストローク病態生理の理解と脳卒中後の神経保護を提供するための新しい治療法の探求を可能にします。光血栓モデルによって生成される小さな皮質ストロークとmicroinfarctionsは、臨床的に無症状または高い有病率を持...

技術はすぐにそのままマウスでローズベンガル光血栓の誘導後のインビボ細胞応答の許可の可視化について説明しました。ローズベンガル（4,5,6,7-テトラクロロ-2 &#39;、4&#39;、5 &#39;、7&#39;-テトラヨード）は、動物モデルにおける虚血性脳卒中（マウス及びラット）を誘導するために使用される感光性染料です。 564 nmのレーザー光を薄く頭蓋骨を介して尾静脈を介して後続の照明RBのボーラス注射に続いて、血栓が生理的発作を引き起こし1に誘導されます。この方法は、もともと1977年にローゼンブラム、エル·Sabbanによって記述し、それ以降の1980年代半ば1,2にワトソンによって適応されました。要するに、ローズベンガルは、その後、組織因子、凝固カスケードの開始剤を活性化し、活性酸素種の生産を生成緑色励起光（この場合は561 nmレーザー）を照射します。凝固カスケードの誘導は虚血性レを生成します臨床ストローク3に病理学的に関連するイオン。 脳卒中は、ニューロン、グリア、内皮および免疫系を含む多くの異なる細胞タイプの相互作用に起因する複雑な病態生理を持っています。特定の細胞プロセスを研究するための最良の方法を選択すると、複数の配慮が必要です。実験技術は、3つのカテゴリのいずれかに広く分類さ： インビトロ 、 インビボ及びそれぞれが長所と短所とインシリコin vitro試験は、それらの天然の環境から細胞を除去する主な欠点を持っているので、そのままで見られる効果を再現できない場合があり、動物を生きた。in vivoでの技術増加翻訳意義と病態の強化実験的複製を提供する。 インシリコ一般的疾患または細胞プロセスのコンピュータモデリングを参照して、ますます試験の潜在的な薬物相互作用を研究するために利用しながら、PLE、収集されたすべての情報は、まだ生きている細胞や組織で試験されなければなりません。 実験室の設定における脳卒中の理想的なモデルは、ヒト集団において見られるものと同様の病理学的特徴を実証する必要があります。ヒト集団において脳卒中の一般的な生理学的特性がありますが、経験した損傷の種類に応じて多くの違いもあります。ヒト集団においてストロークを変化させ、梗塞ボリュームだけでなく、それぞれの病状に関連するメカニズムの違いをもたらすような小さなまたは大血管閉塞、出血性病変、および動脈または心臓塞栓動脈に発生します。動物の脳卒中モデルを利用する利点は、人間のストロークの特性を模倣再現性梗塞の発生です。中大脳動脈閉塞（塞栓または血管内フィラメント法）をモデル遠位MCAOおよび光血栓モデル：もっとも一般的な動物ストロークモデルが使用して動脈閉塞が含まれます。利点各モデルのd欠点は（ 図4、図5を参照してください）他の場所で検討されています。グローバル虚血モデル（MCAO）、実行するのが比較的容易に焦点脳卒中モデルであるよりも、人間のストロークにはあまり関連していました。さらに、これらの方法は、再現性の脳梗塞病変を誘導するのに非常に可変です。実験者は、MCAOモデル上で明確な利点を提供し、よく彼らの実験を制御するように光血栓モデルがあれば高い再現性です。しかし、微小血管傷害のモデルは、最小限虚血性ペナンブラ、細胞を救出6,7であると考えられる領域を表示することが記載されています。また、血管原性浮腫及び細胞毒性浮腫形成はまた、撮像領域の照射後の誘導され得ます。これらの制限にもかかわらず、技術は、脳卒中8、9、10、11以下の多くの生理学的プロセスに新たな洞察を提供しました。

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			<td>330000</td>
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			<td>Optosigma Co</td>
			<td>145-1110</td>
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			<td>Ear bar holder from stereotaxic setup</td>
			<td>Stoelting/Cyborg</td>
			<td>51654</td>
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			<td>DRE, Inc.</td>
			<td>15001</td>
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			<td>DRE, Inc</td>
			<td>86111B</td>
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			<td>Rodent adapter for bain tube</td>
			<td>DRE, Inc</td>
			<td>891000</td>
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			<td>DRE, Inc</td>
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			<td>DRE, Inc</td>
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			<td>134.53</td>
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rose bengal photothrombosis by confocal optical imaging in vivo: a model of single vessel stroke

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to image cellular events in an intact animal. Additionally, the ability to induce physiological disease states such as stroke in vivo increases its utility. The technique described herein allows for physiological assessment of cellular responses within the CNS following a stroke and can be adapted for other pathological conditions being studied. The technique presented uses laser excitation of the photosensitive dye Rose Bengal in vivo to induce a focal ischemic event in a single blood vessel. 
The video protocol demonstrates the preparation of a thin-skulled cranial window over the somatosensory cortex in a mouse for the induction of a Rose Bengal photothrombotic event keeping injury to the underlying dura matter and brain at a minimum. Surgical preparation is initially performed under a dissecting microscope with a custom-made surgical/imaging platform, which is then transferred to a confocal microscope equipped with an inverted objective adaptor. Representative images acquired utilizing this protocol are presented as well as time-lapse sequences of stroke induction. This technique is powerful in that the same area can be imaged repeatedly on subsequent days facilitating longitudinal in vivo studies of pathological processes following stroke.

この方法の目的は、561 nmのレーザー光を薄く頭蓋骨の尾静脈とその後の照明を介してRBのボーラス注射後の動物モデルにおける虚血性脳卒中（マウス、ラット）を誘導することでした。 図4の画像は、0、1、1.5、2分間の領域の照射後の単一の容器内に血栓形成の進行を示しています。血栓形成の前に容器全体はローズベンガルを流れる自由に起因白です。容器の照射誘導後そこに?...

Watch this Scientific Journal Video about Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo:  A Model of Single Vessel Stroke at JoVE.com

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo:  A Model of Single Vessel Stroke

Here, we describe a semi-invasive optical microscopy approach for the induction of a Rose Bengal photothrombotic clot in the somatosensory cortex of a mouse in vivo. The technical aspects of the imaging procedure are described from induction of a photothrombotic event to application and data collection.

共焦点光学イメージングによるローズベンガル光血栓<em&gt;インビボ</em&gt;：単一の容器ストロークのモデル

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to ...

rose-bengal-photothrombosis-confocal-optical-imaging-vivo-model

Research

JoVE Journal

Medicine

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo: A Model of Single Vessel Stroke

14.9K ビュー.  The University of Texas Health Science Center San Antonio. Here, we describe a semi-invasive optical microscopy approach for the induction of a Rose Bengal photothrombotic clot in the somatosensory cortex of a mouse in vivo. The technical aspects of the imaging procedure are described from induction of a photothrombotic event to application and data collection. 

ビデオ: Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo:  A Model of Single Vessel Stroke

Non-invasive Optical Measurement of Cerebral Metabolism and Hemodynamics in Infants

Perinatal brain injury remains a significant cause of infant mortality and morbidity, but there is not yet an effective bedside tool that can accurately screen for brain injury, monitor injury evolution, or assess response to therapy. The energy used by neurons is derived largely from tissue oxidative metabolism, and neural hyperactivity and cell death are reflected by corresponding changes in cerebral oxygen metabolism (CMRO2). Thus, measures of CMRO2 are reflective of neuronal viability and provide critical diagnostic information, making CMRO2 an ideal target for bedside measurement of brain health.
Brain-imaging techniques such as positron emission tomography (PET) and single-photon emission computed tomography (SPECT) yield measures of cerebral glucose and oxygen metabolism, but these techniques require the administration of radionucleotides, so they are used in only the most acute cases.
Continuous-wave near-infrared spectroscopy (CWNIRS) provides non-invasive and non-ionizing radiation measures of hemoglobin oxygen saturation (SO2) as a surrogate for cerebral oxygen consumption. However, SO2 is less than ideal as a surrogate for cerebral oxygen metabolism as it is influenced by both oxygen delivery and consumption. Furthermore, measurements of SO2 are not sensitive enough to detect brain injury hours after the insult 1,2, because oxygen consumption and delivery reach equilibrium after acute transients 3. We investigated the possibility of using more sophisticated NIRS optical methods to quantify cerebral oxygen metabolism at the bedside in healthy and brain-injured newborns. More specifically, we combined the frequency-domain NIRS (FDNIRS) measure of SO2 with the diffuse correlation spectroscopy (DCS) measure of blood flow index (CBFi) to yield an index of CMRO2 (CMRO2i) 4,5.
With the combined FDNIRS/DCS system we are able to quantify cerebral metabolism and hemodynamics. This represents an improvement over CWNIRS for detecting brain health, brain development, and response to therapy in neonates. Moreover, this method adheres to all neonatal intensive care unit (NICU) policies on infection control and institutional policies on laser safety. Future work will seek to integrate the two instruments to reduce acquisition time at the bedside and to implement real-time feedback on data quality to reduce the rate of data rejection.

We combined frequency-domain near-infrared spectroscopy measures of cerebral hemoglobin oxygenation with diffuse correlation spectroscopy measures of cerebral blood flow index to estimate an index of oxygen metabolism. We tested the utility of this measure as a bedside screening tool to evaluate the health and development of the newborn brain.

乳幼児の脳代謝と血行動態の非侵襲的光計測

Perinatal brain injury remains a significant cause of infant mortality  and morbidity, but there is not yet an effective bedside tool that can  accurately ...

医学

In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

Fibered confocal fluorescence in vivo imaging with a fiber optic bundle uses the same principle as fluorescent confocal microscopy. It can excite fluorescent in situ elements through the optical fibers, and then record some of the emitted photons, via the same optical fibers. The light source is a laser that sends the exciting light through an element within the fiber bundle and as it scans over the sample, recreates an image pixel by pixel. As this scan is very fast, by combining it with dedicated image processing software, images in real time with a frequency of 12 frames/sec can be obtained.
We developed a technique to quantitatively characterize capillary morphology and function, using a confocal fluorescence videomicroscopy device. The first step in our experiment was to record 5 sec movies in the four quadrants of the tumor to visualize the capillary network. All movies were processed using software (ImageCell, Mauna Kea Technology, Paris France) that performs an automated segmentation of vessels around a chosen diameter (10 &mu;m in our case). Thus, we could quantify the 'functional capillary density', which is the ratio between the total vessel area and the total area of the image. This parameter was a surrogate marker for microvascular density, usually measured using pathology tools.
The second step was to record movies of the tumor over 20 min to quantify leakage of the macromolecular contrast agent through the capillary wall into the interstitium. By measuring the ratio of signal intensity in the interstitium over that in the vessels, an 'index leakage' was obtained, acting as a surrogate marker for capillary permeability.

In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

In this paper, we present a method to analyze tumor microvessels in vivo using dynamic contrast-enhanced fluorescence videomicroscopy. Two quantitative parameters were acquired: functional capillary density reflecting the vascularity of the tumor, and index leakage reflecting the leakiness of the endothelial wall.

蛍光共焦点ビデオ顕微鏡を使用して腫瘍血管新生のin vivoイメージング

Fibered confocal fluorescence in vivo imaging with a fiber optic bundle uses the same principle as fluorescent confocal microscopy. It can excite ...

A Murine Model of Subarachnoid Hemorrhage

In this video publication a standardized mouse model of subarachnoid hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of Willis perforation (CWp) and proven by intracranial pressure (ICP) monitoring. Thereby a homogenous blood distribution in subarachnoid spaces surrounding the arterial circulation and cerebellar fissures is achieved. Animal physiology is maintained by intubation, mechanical ventilation, and continuous on-line monitoring of various physiological and cardiovascular parameters: body temperature, systemic blood pressure, heart rate, and hemoglobin saturation. Thereby the cerebral perfusion pressure can be tightly monitored resulting in a less variable volume of extravasated blood. This allows a better standardization of endovascular filament perforation in mice and makes the whole model highly reproducible. Thus it is readily available for pharmacological and pathophysiological studies in wild type and genetically altered mice.

A standardized mouse model of subarachnoid hemorrhage by intraluminal Circle of Willis perforation is described. Vessel perforation and subarachnoid bleeding are monitored by intracranial pressure monitoring. In addition various vital parameters are recorded and controlled to maintain physiologic conditions.

くも膜下出血のマウスモデル

In this video publication a standardized mouse model of subarachnoid  hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of  Willis ...

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent marker. Mouse models (brain tumor and arthritis) were used to evaluate the usefulness of this method. Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles tagged with CellVue Maroon (CVM) fluorophore were administered intravenously. Animals were then placed in the rotational holder (MARS) of the in vivo imaging system. Images were acquired in 10&#176; steps over 380&#176;. A rectangular&#160;region of interest (ROI) was placed across the full image width at the model disease site. Within the ROI, and for every image, mean fluorescence intensity was computed after background subtraction. In the mouse models studied, the labeled nanovesicles were taken up in both the orthotopic and transgenic brain tumors, and in the arthritic sites (toes and ankles). Curve analysis of the multi angle image ROIs determined the angle with the highest signal. Thus, the optimal angle for imaging each disease site was characterized. The MAROI method applied to imaging of fluorescent compounds is a noninvasive, economical, and precise tool for in vivo quantitative analysis of the disease states in the described mouse models.

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo quantitation of a fluorescent marker delivered by saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles. Employing mouse models of cancer and arthritis, we demonstrate how the MAROI signal curve analysis can be used for the precise mapping and biological characterization of disease processes.

蛍光SAPC-DOPSナノベシクルを用いて脳腫瘍、関節炎のin vivo光イメージング中

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

脳Microslicesの興奮毒性刺激<em&gt;インビトロ</em&gt;脳卒中のモデル

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Combined In vivo Optical and &#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in vivo optical and &#956;CT imaging allow the visualization of biological phenomena in an anatomical context. These imaging modalities may be especially useful to study conditions that impact bone. In particular, orthopaedic implant infections are an important problem in clinical orthopaedic surgery. These infections are difficult to treat because bacterial biofilms form on the foreign surgically implanted materials, leading to persistent inflammation, osteomyelitis and eventual osteolysis of the bone surrounding the implant, which ultimately results in implant loosening and failure. Here, a mouse model of an infected orthopaedic prosthetic implant was used that involved the surgical placement of a Kirschner-wire implant into an intramedullary canal in the femur in such a way that the end of the implant extended into the knee joint. In this model, LysEGFP mice, a mouse strain that has EGFP-fluorescent neutrophils, were employed in conjunction with a bioluminescent Staphylococcus aureus strain, which naturally emits light. The bacteria were inoculated into the knee joints of the mice prior to closing the surgical site. In vivo bioluminescent and fluorescent imaging was used to quantify the bacterial burden and neutrophil inflammatory response, respectively. In addition, &#956;CT imaging was performed on the same mice so that the 3D location of the bioluminescent and fluorescent optical signals could be co-registered with the anatomical &#956;CT images. To quantify the changes in the bone over time, the outer bone volume of the distal femurs were measured at specific time points using a semi-automated contour based segmentation process. Taken together, the combination of in vivo bioluminescent/fluorescent imaging with &#956;CT imaging may be especially useful for the noninvasive monitoring of the infection, inflammatory response and anatomical changes in bone over time.

Combined In vivo Optical and  &amp;#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Combined optical and &#956;CT imaging in a mouse model of orthopaedic implant infection, utilizing a bioluminescent engineered strain of Staphylococcus aureus, provided the capability to noninvasively and longitudinally monitor the dynamics of the bacterial infection, as well as the corresponding inflammatory response and anatomical changes in the bone.

合わせた<em&gt;インビボ</em&gt;光とμCTイメージングマウスにおける整形外科インプラント感染で感染症、炎症、および骨解剖学を監視する

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in ...

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an increased risk of vascular morbidity.&#160;Yet, albeit&#160;the mounting clinical evidence, there is a paucity of clear studies of vascular toxicity and therefore the etiology of a heterogeneous group of vascular/cardiovascular disorders remains to be elucidated. Moreover, the mechanism that may underlie vascular toxicity can completely differ from the principles of chemotherapy-induced cardiotoxicity, which is related to direct myocyte injury. We have established a real-time, in vivo molecular imaging platform&#160;to evaluate&#160;the potential&#160;acute&#160;vascular toxicity of anti-cancer therapies.
We have set up a platform of in vivo, high-resolution molecular imaging&#160;in mice,&#160;suitable for visualizing vasculature within confined organs and reference blood vessels within the same individuals whereas each individual serve as its own control. Blood vessel walls were impaired after doxorubicin administration, representing a unique mechanism of vascular toxicity that may be the early event in end-organ injury. Herein, the method of fibered confocal fluorescent microscopy (FCFM) based imaging is described, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

We herein describe the method of fibered confocal fluorescent microscopy (FCFM) based imaging, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

化学療法誘発性血管毒性 - リアルタイム<em&gt;インビボ</em船舶減損の&gt;イメージング

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an ...

Photothrombosis-induced Focal Ischemia as a Model of Spinal Cord Injury in Mice

Spinal cord injury (SCI) is a devastating clinical condition causing permanent changes in sensorimotor and autonomic functions of the spinal cord (SC) below the site of injury. The secondary ischemia that develops following the initial mechanical insult is a serious complication of the SCI and severely impairs the function and viability of surviving neuronal and non-neuronal cells in the SC. In addition, ischemia is also responsible for the growth of lesion during chronic phase of injury and interferes with the cellular repair and healing processes. Thus there is a need to develop a spinal cord ischemia model for studying the mechanisms of ischemia-induced pathology. Focal ischemia induced by photothrombosis (PT) is a minimally invasive and very well established procedure used to investigate the pathology of ischemia-induced cell death in the brain. Here, we describe the use of PT to induce an ischemic lesion in the spinal cord of mice. Following retro-orbital sinus injection of Rose Bengal, the posterior spinal vein and other capillaries on the dorsal surface of SC were irradiated with a green light resulting in the formation of a thrombus and thus ischemia in the affected region. Results from histology and immunochemistry studies show that PT-induced ischemia caused spinal cord infarction, loss of neurons and reactive gliosis. Using this technique a highly reproducible and relatively easy model of SCI in mice can be achieved that would serve the purpose of scientific investigations into the mechanisms of ischemia induced cell death as well as the efficacy of neuroprotective drugs. This model will also allow exploration of the pathological changes that occur following SCI in live mice like axonal degeneration and regeneration, neuronal and astrocytic Ca2+ signaling using two-photon microscopy.

Photothrombosis is a minimally invasive and highly reproducible procedure to induce focal ischemia in the spinal cord and serves as a model of spinal cord injury in mice.

マウスの脊髄損傷のモデルとしての光血栓誘発性局所虚血

Spinal cord injury (SCI) is a devastating clinical condition causing permanent changes in sensorimotor and autonomic functions of the spinal cord (SC) ...

Quantitative Visualization of Leukocyte Infiltrate in a Murine Model of Fulminant Myocarditis by Light Sheet Microscopy

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently labelled structures in large biological specimens, and is down to cellular resolution. Meanwhile, the constant refinement of underlying protocols and the enhanced availability of specialized commercial systems enable us to investigate the microstructure of whole mouse organs and even allow for the characterization of cellular behavior in various live-cell imaging approaches. Here, we describe a protocol for the spatial whole-mount visualization and quantification of the CD45+ leukocyte population in inflamed mouse hearts. The method employs a transgenic mouse strain (CD11c.DTR)that has recently been shown to serve as a robust, inducible model for the study of the development of fulminant fatal myocarditis, characterized by lethal cardiac arrhythmias. This protocol includes myocarditis induction, intravital antibody-mediated cell staining, organ preparation, and LSFM with subsequent computer-assisted image post-processing. Although presented as a highly-adapted method for our particular scientific question, the protocol represents the blueprint of an easily adjustable system that can also target completely different fluorescent structures in other organs and even in other species.

Here, we describe a light-sheet microscopy approach to visualize the cardiac CD45+ leukocyte infiltrate in a murine model of aseptic fulminant myocarditis, which is induced by the intratracheal diphtheria toxin treatment of CD11c.DTR mice.

光シート顕微鏡による劇症心筋炎のマウスモデルにおける白血球浸潤の定量的可視化

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently ...

The 4-vessel Sampling Approach to Integrative Studies of Human Placental Physiology In Vivo

The human placenta is highly inaccessible for research while still in utero. The current understanding of human placental physiology in vivo is therefore largely based on animal studies, despite the high diversity among species in placental anatomy, hemodynamics and duration of the pregnancy. The vast majority of human placenta studies are ex vivo perfusion studies or in vitro trophoblast studies. Although in vitro studies and animal models are essential, extrapolation of the results from such studies to the human placenta in vivo is uncertain. We aimed to study human placenta physiology in vivo at term, and present a detailed protocol of the method. Exploiting the intraabdominal access to the uterine vein just before the uterine incision during planned cesarean section, we collect blood samples from the incoming and outgoing vessels on the maternal and fetal sides of the placenta. When combining concentration measurements from blood samples with volume blood flow measurements, we are able to quantify placental and fetal uptake and release of any compound. Furthermore, placental tissue samples from the same mother-fetus pairs can provide measurements of transporter density and activity and other aspects of placental functions in vivo. Through this integrative use of the 4-vessel sampling method we are able to test some of the current concepts of placental nutrient transfer and metabolism in vivo, both in normal and pathological pregnancies. Furthermore, this method enables the identification of substances secreted by the placenta to the maternal circulation, which could be an important contribution to the search for biomarkers of placenta dysfunction.

The 4-vessel Sampling Approach to Integrative Studies of Human Placental Physiology In Vivo

We present a detailed method to study human placental physiology in vivo at term. The method combines blood sampling from the incoming and outgoing vessels on the maternal and fetal sides of the placenta with ultrasound measurements of volume blood flow and placental tissue sampling.

ヒト胎盤生理学の統合研究への4容器サンプリングアプローチ<em&gt;インビボ</em

The human placenta is highly inaccessible for research while still in utero. The current understanding of human placental physiology in vivo is therefore ...